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The Cat in the Hat (Aaron White) acts as the narrator.

In the Jungle of Nool, Horton the Elephant (Nicholas Springer) hears a strange noise coming from a speck of dust. He is convinced there must be someone on it, and rescues the speck and places it on a clover to guard. The other animals (especially the "Sour Kangaroo" (Gwynne Mischler) , a matriarch of the jungle) mock him mercilessly for this, except for Gertrude McFuzz (Stephanie Dietz), who admires his compassion and begins to fall in love with him. However, Horton soon hears the mysterious voice again, which turns out to belong to the Mayor of "Who" (David Riley), a dust-speck planet covered with microscopic inhabitants called Whos. The Cat in the Hat zooms in on the dust speck so the Whos are revealed to the audience. The Mayor of Who and the Whos reveal their planet is so small and fragile they are constantly threatened with death, and beg Horton to help protect them now their cries have reached him. His resolve strengthened, Horton promises he will guard the dust speck, saying "A person's a person, no matter how small."

The mayor's daughter, Jojo (Amanda Wright), is being scolded for thinking "thinks" at school, imaginary thoughts which have apparently disrupted his classes and aroused the ire of her teachers. Although she tries not to think thinks anymore, she starts to imagine her tub is McElligot's pool where her thinks soon spiral out of control and cause time to fly and she goes to bed. Frustrated, the Mayor and his wife agree to "lay down the law" but are unsure of just how to discipline her. After receiving a brochure, they decide to send her to a military school run by a misguided warmonger named General Genghis Khan Schmitz (Aidan Smerud), who is preparing to go to war with those who eat their bread butter side down.

Horton meets Jojo, with whom he bonds instantly as a fellow social outcast. The next day, Gertrude (whose feelings for Horton are still strong) laments that her tail only has one feather, believing its sorry sight is the reason Horton is not noticing her. At the advice of Mayzie La Bird (Emily Hegland), whose tail is enormous and dazzling, Gertrude obtains pills from Doctor Dake (played by the Cat) which Mayzie promises will make her tail "almost as amazing" as hers. Gertrude is so excited about her tail, she overdoses.

Horton is ambushed by a group of mischievous monkeys, the Wickersham Brothers (Thomas Gittens, Alex Grupa, Killian Harnish, Aidan Smerud) who steal the clover and give it to a eagle, Vald Vladikoff (Jacob Sharp), who drops it into a large patch of identical clovers. As Horton begins to look for the clover, Gertrude catches up to him and tries to attract his attention. Horton, however, is so intently focused on his mission he does not notice her. Mayzie LaBird interrupts Horton's search and begs him to sit on it so she can take an afternoon off. Horton reluctantly agrees, and Mayzie flies away, never to return. As winter approaches, Horton is captured by hunters. Gertrude makes an attempt to rescue him, but the size and weight of her new tail prevents her from flying.

Horton is transported to New York City and auctioned off to the circus. Meanwhile, Mayzie is lazing about in Palm Beach.  She decides to attend the Circus, where she reunites with Horton and the egg. Horton pleads for her to take it back, but Mayzie refuses, and gives the egg to Horton to keep forever. Alone with the egg, a sorrowful Horton vows to nurture it himself.

Jojo and her fellow cadets are deployed to fight in the Butter Battle. Jojo finally confronts General Schmitz, declaring his belief in the immorality of the war and deserting the army.

At the Circus, Gertrude reappears and frees a bewildered Horton. She has swallowed her pride, got her tail feathers plucked, and flew across the world to rescue Horton and find the Whos' clover. Ecstatic that the Whos are finally safe, Horton begins to fall in love with Gertrude. However, their happiness is soon crushed as the Sour Kangaroo and the Wickersham Brothers drag Horton back to the Jungle of Nool, and put him on trial for the crimes of "talking to a speck of dust and loitering on an egg". Gertrude and Horton make a stand at the case, but all in vain;  Yertle the Turtle (Nancy Misch) orders the clover to be boiled in a kettle of beezlenut oil. Aghast, Horton begs the Whos to shout to prove their existence, which nearly fails until Jojo comes up with a "think": the word "YOPP" whose peculiar sound is just enough to reach the jungle creatures' ears. The court acquits Horton and the Sour Kangaroo repents, swearing from then on she will help Horton protect the Whos. In Whoville, Jojo is honored as "Thinker Non-Stop" and finally wins the appreciation of his parents.

Suddenly, the egg hatches, and to everyone's surprise a tiny "Elephant-Bird" emerges as a result of Horton having sat on it for so long. Horton panics, realizing he can't handle flying progeny, but Gertrude promises him that as Horton "teaches him earth", she will "teach him sky".